Copyright © 1999, American Society for Microbiology. All Rights Reserved.
A Conventional Beagle Dog Model for Acute and Chronic
Infection with Helicobacter pylori
GIACOMO ROSSI,
1MICHELA ROSSI,
2CLAUDIA G. VITALI,
1DAMIANO FORTUNA,
1DANIELA BURRONI,
2LAURA PANCOTTO,
2SONIA CAPECCHI,
2SABRINA SOZZI,
1GIACOMO RENZONI,
1GIOVANNI BRACA,
1GIUSEPPE DEL GIUDICE,
2RINO RAPPUOLI,
2*
PAOLO GHIARA,
2ANDENNIO TACCINI
1Department of Animal Pathology, Prophylaxis and Food Hygiene, University of Pisa, 50100 Pisa,
1and
IRIS, Chiron SpA Immunobiological Research Institute Siena, 53100 Siena,
2Italy
Received 18 November 1998/Returned for modification 4 February 1999/Accepted 1 March 1999
Helicobacter pylori has been widely recognized as an important human pathogen responsible for chronic
gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Little is
known about the natural history of this infection since patients are usually recognized as having the infection
only after years or decades of chronic disease. Several animal models of H. pylori infection, including those with
different species of rodents, nonhuman primates, and germ-free animals, have been developed. Here we
des-cribe a new animal model in which the clinical, pathological, microbiological, and immunological aspects of
hu-man acute and chronic infection are mimicked and which allows us to monitor these aspects of infection within
the same individuals. Conventional Beagle dogs were infected orally with a mouse-adapted strain of H. pylori and
monitored for up to 24 weeks. Acute infection caused vomiting and diarrhea. The acute phase was followed by
polymorphonuclear cell infiltration, interleukin 8 induction, mononuclear cell recruitment, and the
appear-ance of a specific antibody response against H. pylori. The chronic phase was characterized by gastritis,
epithe-lial alterations, superficial erosions, and the appearance of the typical macroscopic follicles that in humans are
considered possible precursors of MALT lymphoma. In conclusion, infection in this model mimics closely
hu-man infection and allows us to study those phases that cannot be studied in huhu-mans. This new model can be
a unique tool for learning more about the disease and for developing strategies for treatment and prevention.
Chronic infection of human gastroduodenal mucosae by
Helicobacter pylori is associated with chronic gastritis and
pep-tic ulcers and increases the risk of occurrence of gastric
ma-lignancies such as adenocarcinoma and low-grade B-cell
lym-phoma (5, 20, 55, 56). The occurrence of these pathologies
correlates epidemiologically with infection by a particular
sub-set of H. pylori strains, called type I strains (6, 11, 12, 21, 67).
This subset of strains is endowed with increased virulence due
to the expression of a biologically active toxin (VacA), which is
cytopathic to gastric epithelial cells in vitro and in vivo (27, 31,
63), and also due to the acquisition of a pathogenicity island,
called cag, which contains a set of genes encoding several
virulence factors (8) that are involved in the induction of
ty-rosine phosphorylation, pedestal formation, NF-kB activation,
and synthesis of the neutrophil chemotactic cytokine
interleu-kin 8 (IL-8) in gastric epithelial cells (8).
Several animal models of H. pylori infection and disease,
including those with several rodent species and nonhuman
primates, have been proposed. Some of these models also
employ species that are kept under gnotobiotic conditions.
Among these are gnotobiotic piglets (41),
specific-pathogen-free cats (24, 30), gnotobiotic beagle pups (59), and athymic
nu/nu or germ-free mice (39). However, the need to maintain
these animals under germ-free conditions for long periods of
time renders these models impractical, also because they are
technologically sophisticated and particularly expensive.
Fur-thermore, the peculiar immunological status of the gnotobiotic
or immunodeficient hosts employed may jeopardize the
phys-iology of infection and the outcome of the immune response.
More recently, a euthymic, not germ-free, mouse model of
H. pylori infection has been developed. In this model, H. pylori
freshly isolated from human gastroduodenal biopsies have
been adapted to persistently colonize the gastric mucosae of
mice (47). This model has proven particularly useful for
as-sessing the feasibility of either preventive (46–48, 58) or
ther-apeutic (28) vaccination, as well as for the in vivo screening of
anti-H. pylori antimicrobials (43) and for studying the
patho-genesis of infection (22, 60). However, infected mice do not
develop symptoms and they need to be sacrificed in order to
evaluate gastric infection. Thus, the pathological changes
in-duced by chronic infection and/or the effects of therapeutic or
immunizing regimens cannot be followed up in the same
indi-vidual.
A more physiologically relevant animal model in which
in-fection resembles closely human H. pylori inin-fection would
cer-tainly be desirable. Nonhuman primates have been proposed
as a model of experimental infection with H. pylori. However,
several factors, including cost and housing, do not allow the
extensive application of this model. Furthermore, most of
these monkeys (e.g., rhesus monkeys) are usually
spontane-ously infected with Helicobacter (19).
Conventional beagle dogs have already been used to
repro-duce experimental infections with human pathogens such as
Yersinia enterocolitica (32), Borrelia burgdorferi (9), and
Leish-mania infantum (4). Furthermore, it has been reported that the
gastroduodenal mucosae of conventional dogs may be
natu-rally colonized by some gastrospirilla (35, 36), which may
oc-casionally cause mild gastritis, but not by H. pylori (36).
In this study we have assessed the feasibility of establishing
* Corresponding author. Mailing address: Chiron SpA
Immunobio-logical Research Institute Siena, Via Fiorentina 1, 53100 Siena, Italy.
Phone: 39-0577-243414. Fax: 39-0577-243564. E-mail: rappuoli@iris02
.biocine.it.
3112
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H. pylori infection in conventional dogs, using a strain of H.
py-lori previously adapted to the mouse (47). We report that
H. pylori can colonize the gastric mucosae of conventional
beagle dogs, causing both acute symptoms and long-term
chronic infection. The animal model described here is unique
because it is the only model in which the animals show acute
symptoms that resemble some of those described during
ex-perimental infection of humans.
MATERIALS AND METHODS
H. pylori strain.SPM326s, a streptomycin-resistant derivative of the mouse-adapted H. pylori type I (CagA1VacA1) strain SPM326 (47), was obtained by allelic exchange of the S12 gene with a mutated gene sequence harbored by a naturally occurring streptomycin-resistant H. pylori strain. This strain has been shown to be as infective and virulent in mice as its parental strain. Details of the experimental procedure followed to obtain this strain and of the strain’s infec-tivity in mice will be described elsewhere (47a).
Animals and experimental design.Three 4- to 6-month-old conventional bea-gle dogs, one male and two females (Morini SpA, San Polo D’Enza, Reggio Emilia, Italy), were selected on the basis of the absence of detectable immuno-globulin G (IgG) against H. pylori in serum in Western blot (WB) analysis with total bacterial lysate as the antigen (see below). The three dogs selected were housed under standard conditions and maintained on a diet of dry food (MIL; Morini SpA) and tap water ad libitum. Upon arrival of the dogs in our animal facilities, an additional WB analysis of sera confirmed their H. pylori status. The dogs were housed in individual boxes and allowed to adapt for a month to their new environment. During the month of adaptation, two tests were carried out on fecal samples to assess the possible presence of intestinal parasites or common enteric pathogenic bacteria.
The dogs were then challenged every other day over a 1-week period, for a total of three times, with the mouse-adapted strain H. pylori SPM326s as follows. Twenty-four hours before each challenge the dogs were not given food. Two hours before bacterial inoculation the dogs received 10 mg of cimetidine (Taga-met 200; Smith Kline & French, Philadelphia, Pa.) intramuscularly per kg of body weight. At the moment of challenge, the dogs were intravenously anesthe-tized with a mixture of 40mg of medetomidine chloridrate (Domitor; Centralvet-Vetem SpA, Milano, Italy) per kg and 5 mg of ketamine (Ketavet; Gellini, Latina, Italy) per kg, and then a gastric lavage was performed with 100 ml of sterile 0.2 M NaHCO3, followed by oral challenge with 3 ml of a freshly prepared suspension in sterile saline of 109CFU of H. pylori SPM326s, grown under microaerobic conditions (see below). At the end of the bacterial inoculation, 200 mg of the anesthetic antagonist atipamezole (Antisedan; Centralvet-Vetem SpA) was administered, and then dogs were again treated with cimetidine and fed after 2 h.
Dogs were observed daily for the appearance of any signs, with particular attention being paid to gastrointestinal signs, such as vomiting and diarrhea. Prior to endoscopic examinations, blood samples, salivary samples, and oropha-ryngeal and rectal swabs were collected under sterile conditions immediately before the challenge and then at 1, 2, 4, 8, 12, 18, and 24 weeks. At the same times gastric endoscopy was performed, following anesthesia carried out as described above, with a 4.9-mm-diameter pediatric bronchoscope (Pentax Tech-nologies, Zaventem, Belgium). Gastric biopsies were taken during the endoscopy with flexible pinch biopsy forceps at the antrum, corpus, fundus, and cardia before the challenge for urease testing and for microbiological, PCR, histopatho-logical, and immunohistochemical analyses. Before each endoscopy the whole instrument and the flexible forceps were soaked in 4% glutaraldehyde for 45 min and then rinsed in sterile saline. To avoid cross-contamination among biopsies taken at different sites, the forceps were washed with tap water and lightly flame sterilized before the collection of each biopsy sample.
In a second experiment, three 4- to 6-month-old female beagle dogs were infected and followed up as described above; three additional dogs were kept uninfected, as a control, for at least 5 months. Also in this experiment, dogs were housed in single boxes.
The experimental protocol was approved by the Scientific and Ethical Com-mittee of the University of Pisa and received official authorization by the Italian Ministry of Health (Department of Veterinary Health, Food and Nutrition, DM no. 21/97-C).
Rapid urease test.Antral biopsies were incubated for up to 24 h in 1 ml of a 10% urea solution in distilled water to which 2 drops of a 1% phenol red solution (Sigma) in sodium phosphate buffer (pH 6.5) had been added. A positive test is indicated by change of color (from orange to dark pink) in the medium; the time necessary for the color change is recorded. The time to positivity of this test has been shown to be proportional to the number of bacteria present at the biopsy site (33).
Histopathology and transmission electron microscopy (TEM).Samples for histological, immunohistochemical, and ultrastructural examinations were taken from the biopsies at sites adjacent to those used for microbiological analysis and for PCR. The samples were fixed in 10% buffered formalin and embedded in
paraffin. Three-micrometer-thick sections were stained with hematoxylin-eosin (HE) and Alcian and periodic acid-Schiff (PAS) stain by standard procedures for histopathological examination.
Similar sections were also employed for immunohistochemical analyses by the ABC-peroxidase technique with a monoclonal antibody (MAb) specific for H.
py-lori (Biogenesis Ltd., Poole, England), a mouse MAb specific for VacA (clone
C1G9) obtained by immunizing BALB/c mice with purified native H. pylori VacA (7a), or a mouse MAb specific for human IL-8 (clone DM/C7; Genzyme Diag-nostics, Cambridge, Mass.). For IL-8 detection, sections were placed onto pre-treated slides (Bio-Optica, Milan, Italy) to promote adhesion and dried over-night at 37°C. After being dewaxed, sections were immersed in citrate buffer (10 mM, pH 6.0) and processed in a microwave oven at 650 W for 10 min. Slides were then allowed to cool at room temperature for at least 20 min before being further processed for immunostaining by standard procedures. Biotinylated horse anti-mouse antibody (Dako, Milan, Italy) was used as secondary antibody. The reac-tion was developed with 3-1-diaminobenzidine-chlorhydrate (Sigma Chemical Co., St. Louis, Mo.) for the identification and localization of bacterial antigen and for the detection of IL-8.
For electron microscopy, samples were fixed in Karnowsky, postfixed in OsO4, and embedded in Epon-Araldite (Polysciences Inc., Warrington, Pa.). Semithin sections were stained with toluidine blue for evaluation of cell damage, whereas ultrathin sections were stained with uranyl acetate and lead citrate and then examined with a model EM 301 TEM (Philips, Eindhoven, The Netherlands) operating at 80 kV.
Bacterial growth conditions.Gastric biopsies taken from the different gastric sites, as well as specimens from rectal and oropharyngeal swabs, were streaked onto Columbia agar plates (Gibco BRL, Paisley, United Kingdom) containing 5% horse blood supplemented with Dent’s or Skirrow’s antibiotic mixture (Ox-oid, Basingstoke, United Kingdom) plus 400mg of streptomycin (Sigma) per ml. Plates were incubated under microaerobic conditions with an Anaerojar system and a Campygen atmosphere-generating system (Oxoid) for 7 to 12 days. Grow-ing H. pylori colonies were identified by their typical morphology, as assessed by visual inspection of the plates, and then confirmed by Gram staining, urease testing, and PCR amplification of the cagA gene.
PCR.Bacteria or gastric biopsy samples were incubated at 37°C for 30 min with lysozyme at 100mg/ml in 0.1 M NaCl–1 mM EDTA–10 mM Tris-HCl (pH 8). Then sodium dodecyl sulfate was added (at a final concentration of 1%, wt/vol), and the mixture was heated to 65°C. Proteinase K (25mg/ml) was added before incubation at 50°C for 2 h. Then the mixture was extracted with phenol-chloroform. After extraction, the DNA solution was mixed with absolute ethanol at220°C for 1 h, washed with 75% ethanol, and dried. The DNA pellet was suspended in distilled water (11).
Oligonucleotides were synthesized on an automatic synthesizer (Applied Bio-systems Inc., Foster City, Calif.) by the automated phosphoramidite coupling method and purified by standard protocols. The H. pylori CagA primers R008 (59-TTAGAATAATCAACAAACATCACGCCAT-39) and D008 (59-AATGCT AAATTAGACAACTTGAGCGA-39) were derived from the sequenced cagA gene (11), giving an amplified product of 298 bp. The PCR mixture contained 50 mM KCl, 10 mM Tris, 200 mM deoxynucleoside triphosphates, 30 pmol of each primer, 0.1mg of bovine serum albumin, 2.5 U of AmpliTaq (Perkin-Elmer, Norwalk, Conn.), and 80 ng of H. pylori chromosomal DNA. Amplifications were performed on a PCR system 9600 thermocycler (Perkin-Elmer) with the follow-ing cyclfollow-ing profile: 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s for 38 cycles and then extension at 72°C for 7 min.
Detection of anti-H. pylori antibodies.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H. pylori (strain SPM326s) and WB analysis of sera were performed according to previously published procedures (47). Briefly, dog sera were diluted 1:200 and incubated for 2 h at room temperature. Then, horseradish peroxidase-conjugated rabbit anti-dog IgG antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) was added at a 1:2,000 dilution for 2 h and the reaction mixture was developed with 4-a-chloronaphtol as the substrate. Detection of antibody against H. pylori by enzyme-linked immunosorbent assay was carried out on 96-well plates coated overnight at 4°C with SPM326s lysate (10mg/well) or with purified native VacA or CagA (0.2 mg/well). Coated wells were blocked with phosphate-buffered saline containing 5% nonfat milk. Two-fold serial dilutions of the sera were incubated at 37°C for 2 h and then washed with phosphate-buffered saline. Antigen-specific IgG, IgG1, and IgG2 titers were determined with appropriate dilutions of horseradish peroxidase-conjugated rab-bit anti-dog IgG (Nordic), goat anti-dog IgG1, or sheep anti-dog IgG2 (Bethyl Laboratories Inc., Montgomery, Tex.) polyclonal antibody for 2 h at 37°C. An-tigen-bound antibodies were revealed by adding o-phenylenediamine dihydro-chloride (Sigma) as a substrate. Antibody titers were determined as previously described (28).
For the detection of H. pylori-specific antibodies in the saliva, we used a similar procedure and an SPM326s lysate (10mg/well) to coat the plates. Specific alka-line-phosphatase-conjugated anti-dog IgG and IgA antibodies (Nordic) were added at an appropriate dilution. Results were expressed as optical densities at 405 nm of the salivary samples tested at a 1:20 dilution.
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RESULTS
Acute symptoms following H. pylori infection.
Three
conven-tional beagle dogs were inoculated three times with 10
9CFU
of a virulent H. pylori strain and then closely followed up for
the appearance of signs and for histopathological lesions over
time. During the first week after the third challenge with H.
py-lori, the three dogs experienced at least one episode per day of
loose stools, a condition which persisted, but occurred less
frequently, in the second week. Furthermore, during the first
week postinfection (p.i.), the dogs had episodes of mucous and
foamy vomiting, often containing gastric juices and not
asso-ciated with food intake. These symptoms disappeared
sponta-neously without specific treatment, and no other symptoms
were observed. All three dogs remained alert and ate normally
throughout the whole study. The same symptoms appeared in
two of the three dogs infected in the second experiment and
again disappeared spontaneously. Uninfected control dogs did
not show any vomiting or diarrhea.
Histopathology and endoscopic findings.
Results of
endo-scopic and histological examinations of the gastric mucosae
performed at time zero were normal for all the dogs used in the
two experiments (not shown). In the histological sections of
biopsies taken at 1 week p.i. we observed a marked hyperemia
and edema of the mucosal lamina propria, particularly in the
corpus and in the antrum (Fig. 1A). At this time acute antral
gastritis was evident and was characterized histologically by the
presence of numerous polymorphonuclear leukocytes
infiltrat-ing the laminae propriae among the glands, the structure of
which was in some cases significantly altered (Fig. 1B).
Nota-bly, several polymorphonuclear leukocytes were interspersed
in the epithelial cell layer and were also found in the mucus
(not shown), suggesting that neutrophil transcytosis was
elic-ited by the infection.
Upon endoscopic examination performed 1 and 2 weeks p.i.,
a gastric hyperemia was observed in all infected dogs. Edema
of the mucosa and catarrh adhering to the mucosae of the
antrum and corpus were present, conferring a congested aspect
to the gastric wall. At 2 weeks p.i., histology of the gastric
biopsies evidenced a decrease in the level of neutrophilic
in-filtration into the lamina propria and a concomitant increase in
the level of mononuclear cells. Edema and hyperemia of the
lamina propria were still present (not shown).
After 4 weeks, clear signs of superficial erosions were
endo-scopically evident in the antrum. Histological sections showed
vacuolar degeneration, loss of the apical secreting portion of
the cells, piknosis, and rhexis of epithelial cell nuclei (Fig. 1C
to E).
At 8 weeks p.i., chronic follicular gastritis, with the
charac-teristic “bumpy” aspect of the gastric wall, was easily detected
by endoscopy in the infected animals. Histological examination
of gastric biopsies revealed the presence of small
lymphoplas-macytic aggregates among the glands of the corpus and fundus;
lymphoid follicles (1.5 to 2 mm) appeared in the antrum (Fig.
1F). Degenerative processes in the epithelial cells, the
pres-ence of scattered neutrophilic granulocytes, and an increase in
the exocytosis of mononuclear cells were associated with these
follicles. All these signs have been reported as being typical of
chronic active gastritis in infected humans (13, 26, 29, 54, 62,
66). These macroscopic and microscopic signs remained
un-changed at later times (i.e., at 12, 18, and 24 weeks p.i.). At this
advanced stage of infection, marked modifications in the
mu-cin composition, as detected by Alcian-PAS staining of
histo-logical sections, was also observed, with a progressive
deple-tion of PAS-positive cells in the antrum, suggestive of a
functional gastric atrophy (not shown).
H. pylori was identified in the biopsies
immunohistochemi-cally with the C1G9 MAb specific for VacA. H. pylori was also
observed by TEM in the lumens of antral glands, in close
contact with the luminal surfaces of epithelial cells (Fig. 2).
Control dogs, which were not infected, always showed
nor-mal mucosae upon endoscopical, histological, and
immunohis-tochemical examination for the entire period of the follow-up.
Immunohistochemical detection of IL-8 in gastric biopsies.
One of the most striking findings of the present work was the
demonstration of a strong infiltration of neutrophils within the
lamina propria and the superficial epithelia during the first
weeks of infection, which then became much less evident. To
investigate whether this early infiltration was mediated by IL-8,
as reported for humans (14), the presence of this chemokine
was investigated immunohistochemically. Histological sections
from biopsies taken before infection were consistently negative
for the presence of IL-8. At the first and second weeks after
infection, IL-8 was expressed by gastric epithelial cells,
partic-ularly in areas corresponding to a more pronounced
neutro-philic infiltration in the underlying lamina propria or in areas
where neutrophil transcytosis was more detectable (Fig. 3).
IL-8-positive mononuclear cells were also found interspersed
in the lamina propria in correspondence with neutrophilic
in-filtrates (not shown). IL-8 became undetectable in sections
from biopsies taken 8 weeks after infection (Fig. 3B), at a time
when neutrophilic infiltration was much less prominent than in
the early phases of infection.
Isolation and identification of H. pylori.
The presence of
H. pylori in the biopsies taken from the gastric mucosae of
experimentally infected dogs was first assessed by the rapid
urease test. The test, which gave negative results for the gastric
biopsies taken before the oral challenge with H. pylori and for
samples from the control, uninfected dogs, showed strongly
and quickly positive results (a few minutes to 2 h) for the
infected dogs in the two experiments from the first sampling, 1
week after infection, and continued to show strongly positive
results at all time points during the follow-up of the study.
Antral biopsies from each dog were also applied to
micro-biological culture for H. pylori identification. In samples taken
1, 2, and 4 weeks p.i., H. pylori was identified by culture,
although at these time points the growth of bacteria from the
selective culture plates was rather scanty. From the eighth
week onward, the growth of H. pylori from all the gastric biopsy
samples was more evident, and 50 to 200 H. pylori colonies
were easily recovered from each biopsy. At later times (i.e., at
18 and 24 weeks) the bacterium was easily isolated from the
corpus and antrum only, whereas a marked decrease in the
growth efficiency was observed in biopsies taken from cardias
and fundi.
Starting from the first week p.i., H. pylori could also be
cultured from rectal swabs. These microbiological findings
were always confirmed by PCR performed on the bacterial
colonies grown in the plates.
PCR was carried out on antral biopsy samples taken at time
zero and at each endoscopic examination with cagA-specific
primers. The presence of H. pylori was confirmed in all p.i.
samples. Figure 4A shows the PCR products that confirmed
the presence of H. pylori in antral gastric biopsies taken at 4
and 12 weeks p.i. from each of the three dogs of the first
experiment. Although all attempts to culture H. pylori from
oropharyngeal swab specimens were unsuccessful at any time
during the follow-up, H. pylori DNA was detectable by PCR
amplification of the cagA gene at all times p.i., even 24 weeks
p.i. (Fig. 4B).
Anti-H. pylori antibody response.
The induction of a specific
H. pylori antibody response was followed up with serum and
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FIG. 1. Histopathological findings with infected dogs. (A) HE stain of gastric mucosa at 1 week p.i. showing hyperemia and edema of the mucosal lamina propria (bar5 25 mm); (B) HE staining of antral mucosa at 1 week p.i. showing numerous polymorphonuclear granulocytes infiltrating the laminae propriae among the glands (bar5 25 mm); (C) HE stain of antral mucosa at 4 weeks p.i. showing vacuolar degeneration of the epithelium (bar 5 25 mm); (D) evident epithelium damage in the antral mucosa at 4 weeks p.i. after toluidine blue staining of a semithin section (bar5 100 mm); (E) high-power magnification (bar 5 12.5 mm) of the field in panel D showing some curved bacteria (arrows) close to epithelial cells which are heavily vacuolized and show loss of the apical secreting portion; (F) HE stain of a biopsy taken 8 weeks p.i. showing a characteristic lymphoplasmacytic follicle associated with chorion edema and displacement of glands (bar5 400 mm).
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salivary samples taken before and at different times after
in-fection and analyzed by enzyme-linked immunosorbent assay.
Infected dogs mounted a fast serum IgG antibody response to
H. pylori antigens when we used both a crude bacterial lysate
(Fig. 5A) and purified recombinant CagA and VacA (not
shown). This antibody response was already easily detectable
in the serum samples 2 to 4 weeks after infection and persisted
for the entire period of the study. It is noteworthy that most of
the H. pylori-specific IgG in serum belonged to the IgG2
iso-type, with IgG1 being undetectable for the entire follow-up of
the study (Fig. 5A). Furthermore, and interestingly, both IgA
and IgG antibodies binding to H. pylori lysate were also
de-tected in the samples of saliva collected after infection (Fig.
5B). In the saliva, H. pylori-specific antibodies exhibited a
slower kinetic compared to that observed in the sera. In fact,
salivary IgA and IgG reached their peaks 12 to 15 weeks after
infection and then declined, although they were still detectable
at 24 weeks.
DISCUSSION
The increasing importance of H. pylori in the induction of a
wide variety of gastric pathologies has stressed the need for a
physiological animal model to test the pathophysiology of the
infection and to aid in the development of new drugs and
vaccines. In this paper we describe the experimental infection
of conventional dogs with H. pylori and the long-term outcome
of this infection. We show that a mouse-adapted H. pylori type
I strain persistently colonizes the stomachs of conventional
beagle dogs, causes acute symptoms and gastric pathology, and
elicits specific immune responses.
A remarkable feature of the model described here is that the
infected dogs presented evident clinical signs, such as vomiting
and diarrhea, at the time of the onset of infection, which lasted
for 1 to 2 weeks. This is the first animal model in which acute
symptoms were observed as a consequence of gastric
inocula-tion of H. pylori. In a study carried out with 7-day-old
gnoto-biotic beagle pups, Radin et al. (59) did not report any acute
symptoms, with the pups remaining asymptomatic for the
du-ration (30 days) of the experiment. There may be several
ex-planations for the occurrence of evident clinical signs in our
study. First, in the present study we used a phenotypically
characterized cytotoxic type I strain which had been adapted to
a nonhuman host by several in vivo passages. Second, we used
a larger dose of bacteria (three inocula of 10
9bacteria) than
that (one single bolus of 3
3 10
8bacteria) employed by Radin
et al. (59). Third, the animals used in our study had a normal
gastrointestinal commensal microbial flora, which may have
contributed to amplification of the pathological consequences
of infection. The possibility that the acute symptoms were
induced by the experimental manipulation of the dogs (i.e.,
anesthesia, endoscopy, etc.) is highly unlikely because
unin-fected control dogs did not show any symptom, despite the fact
that they were subjected to the same treatments as infected
dogs. Furthermore, these symptoms were never seen at later
stages of infection.
We believe that infection of conventional beagle dogs
repro-duces the human situation and allows us to study also the acute
phase, a step that is never noticed in humans and is not
repro-duced in any of the other animal models. Although the natural
history of H. pylori infection is poorly known and, as a
conse-quence, the documentation of the clinical picture of the early
stages of acute infection in humans is scarce (49, 51, 53, 61), it
is noteworthy that in self-infection experiments, vomiting (49,
53), as well as an increase in intestinal peristalsis and a
tran-sient softening of feces (49), has been reported. Furthermore,
VacA toxic activity was also found in a proportion of diarrheal
feces from children (44, 45). It is then logical to hypothesize
that the acute symptoms observed in the dogs of the present
study may reproduce those which can be encountered during
the early stages of H. pylori infection in humans.
One of the advantages of the conventional dog model
pre-sented here is the ease of follow-up of the infection over a long
period by endoscopy, at variance with small animal models and
with the time limitations imposed by the germ-free animal
models (59). This advantage has been exploited in the present
investigation, where it was possible to follow up for at least 24
weeks the occurrence and progression of histopathological
le-sions induced by the infection.
Indeed, the dogs developed early superficial gastritis, with
the appearance of mucosal erosions, which progressed to
fol-licular gastritis. It is remarkable that at early stages of infection
the gastric lamina propria was heavily infiltrated by
polymor-phonuclear cells. In other H. pylori animal models, this
neu-trophil infiltration is usually much less pronounced (41, 47) or
has been described only at later stages of infection (42, 59).
This infiltration, accompanied by symptoms like vomiting,
mimics that observed in infected humans with acute gastritis
(49, 53, 61), which has been hypothesized to be mediated by
proinflammatory chemokines, such as IL-8, specifically
in-duced by products of cytotoxic (type I) H. pylori strains (2, 14,
38, 57, 65).
In the present study, where we used a well-characterized
type I strain to infect dogs, IL-8 could be easily detected by
FIG. 2. TEM showing the ultrastructural aspect of antral mucosa 18 weeksp.i. Typical H. pylori bacteria are visible in a glandular lumen. Magnification,
37,920.
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immunohistochemistry in early stages of infection.
Interest-ingly, the time course of expression of this chemokine
paral-leled that of infiltration by neutrophils. In fact, it was more
evident when neutrophils were more abundant in the lamina
propria and rapidly decreased at later stages, when the
infil-trates were more characterized by mononuclear cells. The
de-tection of IL-8 in the gastric epithelia of infected dogs with a
MAb directed against human IL-8 was clearly not due to
non-specific binding. In fact, human IL-8 and dog IL-8 have 75%
identity both at the nucleotide and at the amino acid level (50).
Furthermore, the absence of detectable positivity for IL-8 in
sections taken 8 weeks p.i. speaks in favor of the specificity of
our results. Previous studies of humans have clearly shown that
IL-8 can be detected immunochemically (14) and
immunohis-tochemically (15) in gastric biopsies from individuals infected
with H. pylori. In humans, however, gastric epithelia from
nor-mal, noninfected individuals can be reactive with anti-IL-8
antibodies (15). In the present study, normal gastric mucosa
was always negative. This difference may be explained by the
fact that most of the human studies are carried out with adult
individuals, in which IL-8 induction may be mediated by
exog-enous (e.g., bacterial lipopolysaccharide) and/or endogexog-enous
factors not related to H. pylori. On the contrary, in this study,
we used young (4- to 6-month-old) conventional beagle dogs,
which had had less time and opportunity to have contacts with
other IL-8-inducing factors. On the other hand, a recent study
FIG. 3. Detection of IL-8 expression by immunohistochemistry. (A and B) Representative fields of sections obtained from antral biopsies collected at 2 and 8 weeks p.i., respectively. Sections were stained with anti-IL-8 MAb and counterstained with hematoxylin. In panel A, IL-8-positive epithelial cells are indicated by arrows. Magnification,3100.on May 9, 2021 by guest
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has clearly shown that 1 month after successful eradication of
H. pylori infection patients experience a significant reduction of
infiltrating neutrophils and normalization of the mucosal IL-8
levels (1). These data are in full agreement with the kinetics of
appearance of neutrophil infiltration and IL-8 detection in the
gastric mucosae of infected dogs in the present study.
With the progression of infection, clear signs of epithelial
erosions appeared both macroscopically and microscopically,
with characteristic cellular vacuolization and loss of the apical
portions of the epithelial cells. We have previously observed
similar lesions in the gastric mucosae of H. pylori-infected mice
(28, 47). One of the most striking features at later stages of
infection was the presence of well-structured lymphoid follicles
both in the corpus and in the antrum. This is a finding typical
of chronic stages of infection by Helicobacter species and has
been extensively reported both for chronically infected patients
(26, 54) and for experimentally infected animals (24, 28, 30,
59). These organized lymphoid structures may be the
conse-quence of an active and persistent stimulation of the immune
system at the local mucosal level, with recruitment in situ of
specific T lymphocytes (37). It has been suggested that in some
individuals such lymphoid structures in the gastric mucosa may
precede the development of low-grade B-cell lymphoma (56).
More recently this hypothesis has been proven by showing
B-cell clonality at the site of chronic gastritis in patients with
chronic H. pylori gastritis and subsequent gastric
mucosa-asso-ciated lymphoid tissue lymphoma (68). Thus, the present
model of chronic infection with H. pylori may turn out to be
useful also in investigating the kinetics of the organization of
lymphoid follicles in the gastric mucosa and eventually in
test-ing the development of gastric cancer.
During the entire follow-up, the persistence of gastric
colo-nization by H. pylori in the gastric biopsies was assessed by
urease testing, PCR, immunohistochemistry, and TEM.
Fur-thermore, it was also possible to culture the bacteria from the
gastric biopsies. These facts clearly show that the evolution of
the pathology in this model is constantly associated with the
presence of H. pylori. It should be stressed that viable H. pylori
was also recovered from rectal swabs at every time of
obser-vation, even at 24 weeks after infection. The ability to culture
strain SPM326s from the fecal swabs of conventional animals is
likely to be a consequence of the fact that, since this strain
bears a gene for streptomycin resistance, it is possible to
effi-ciently use selective culture conditions to isolate it from
con-taminating commensal flora normally present in these samples.
Bacterial isolation from feces has been reported for
experi-mental Helicobacter mustelae infection of ferrets (25). These
findings demonstrate that, at least in some animal models, a
considerable quantity of viable H. pylori organisms is
elimi-nated in the feces. Should this be applicable to human
infec-tions, as claimed by some researchers (40, 64), it is tempting to
hypothesize that infected individuals can spread viable H.
py-lori with stools and that, under certain epidemiological
condi-tions (3, 34), this may represent a source of dissemination to
other subjects. It is, however, highly unlikely that oral-fecal
transmission caused the persistence of the chronic infection in
our experiments, since the dogs were constantly kept under
isolation in separated individual boxes. In fact, control dogs,
kept under the same conditions, never exhibited any signs of
infection with H. pylori.
Our data show that H. pylori DNA could also be detected in
the oropharyngeal swabs of infected dogs during the entire
follow-up. The presence of bacterial DNA was not due to
contamination from endoscopic manipulation, since all
sam-FIG. 4. Detection of H. pylori in gastric biopsies and oropharyngeal swabspecimens by PCR. (A) H. pylori DNA amplification of the cagA sequence showing the expected PCR product of 298 bp in gastric biopsies of dogs collected at 4 and 12 weeks p.i. but not in gastric samples collected before infection. (B)
cagA amplification obtained in material extracted from oropharyngeal swab
specimens of dogs taken at 0, 4, and 24 weeks. Lanes: M, markers; PC, positive controls; W, water.
FIG. 5. Anti-H. pylori antibody responses in infected dogs. (A) Serum IgG and IgG subclass antibody responses to H. pylori lysate. Each curve represents the mean titers at the different time points. (B) Salivary IgA and IgG antibody responses to H. pylori lysate. Each curve represents the mean titers at the different time points obtained with salivary samples diluted 1:20.
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ples, including orophraryngeal swab specimens, were taken
before endoscopic examination, or a result of vomiting, since
specific PCR products were still detectable 24 weeks after
infection. It is possible that H. pylori or H. pylori material
reaches the oral cavity through episodes of gastroesophageal
regurgitation. Nevertheless, since it was not possible in the
present study to isolate viable (i.e., culturable) forms of the
bacterium, it is not possible to conclude whether this might
represent another possible route of transmission of infection to
other individuals, as has been suggested by others (3).
The experimental infection with H. pylori induced early
de-tectable antibody responses to different antigens, including
CagA and VacA, which persisted during the entire period of
follow-up. Interestingly, the majority of the anti-H. pylori IgG
antibodies detected in the serum belonged to the IgG2 isotype.
A predominant production of serum IgG2 has been reported
for dogs in association with chronic infections, such as with
Toxoplasma gondii and Leishmania infantum (7, 18), whereas
preferential production of IgG1 has been associated with
hel-minthic infections or allergies (16, 18, 62). Our data, thus,
suggest that experimental H. pylori infection in dogs is
associ-ated with a preferential activation of Th1-type CD4
1T-cell
subsets, in agreement with data previously obtained with mice
(28, 52) and humans (17). It is remarkable that infected dogs
exhibited H. pylori-specific IgA and IgG antibody responses in
their saliva, which peaked 12 to 15 weeks after infection. Taken
together, these data clearly show that experimental infection of
conventional dogs with H. pylori induces specific
seroconver-sion and mucosal (salivary) antibody response. Once again,
these findings are in full agreement with those amply reported
for humans in terms of both serum (20) and salivary antibodies
(10, 23) specifically induced after infection with H. pylori.
In conclusion, the H. pylori infection of conventional dogs
described here mimics clinical, pathological, microbiological,
and serological aspects of the infection in humans. In fact,
acute infection induces symptoms (vomiting and diarrhea) that
disappear spontaneously and acute gastritis, with early
recruit-ment of neutrophils, possibly mediated by H. pylori-induced
IL-8, followed by the appearance of superficial erosion and of
lymphoid follicles and chronic gastritis. Bacterial colonization
is chronic, inducing an early and sustained immune response.
Unlike most of the animal models of H. pylori infection, in
which animals are sacrificed at given time points to evaluate
both infection and pathology, this model has allowed us to
monitor the evolution of the consequences of H. pylori
infec-tion at both acute and chronic stages in the same individuals. It
is likely that this animal model will permit a better
investiga-tion of the pathogenesis of the bacterium and will also allow us
to better assess the feasibility of vaccination. In particular, it
will now be possible to investigate the effect of both preventive
and therapeutic vaccination regimens on the evolution of
in-fection and disease in the same individual.
ACKNOWLEDGMENTS
We thank A. Covacci and M. Marchetti for H. pylori SPM326s, M.
Carlucci for revising the manuscript, and N. Figura and S. Censini for
helpful advice. We are also grateful to V. Mazzoncini (Abiogen
Pharma, Migliarino Pisano, Pisa, Italy) for kindly providing the
hous-ing for the dogs used in this study and A. Fornaciari (Morini SpA) for
allowing us free access to the breeding of the beagle dogs for the
selection of the dogs used in this study.
E.T. was supported in part by the University of Pisa (Fondi di
Ateneo).
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